Safety Assessment of Plant- Derived and Peptides as Used in Cosmetics

Status: Scientific Literature Review for Public Comment Release Date: October 13, 2016 Panel Meeting Date: December 5-6, 2016

All interested persons are provided 60 days from the above date to comment on this safety assessment and to identify additional published data that should be included or provide unpublished data which can be made public and included. Information may be submitted without identifying the source or the trade name of the cosmetic product containing the ingredient. All unpublished data submitted to CIR will be discussed in open meetings, will be available at the CIR office for review by any interested party and may be cited in a peer-reviewed scientific journal. Please submit data, comments, or requests to the CIR Director, Dr. Lillian J. Gill.

The 2016 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is Lillian J. Gill, DPA. This safety assessment was prepared by Christina L. Burnett, Scientific Analyst/Writer and Bart Heldreth, Ph.D., Chemist CIR.

© Cosmetic Ingredient Review 1620 L St NW, Suite 1200◊ Washington, DC 20036-4702 ◊ ph 202.331.0651 ◊fax 202.331.0088 ◊ [email protected] INTRODUCTION The plant-derived proteins and peptides detailed in this report are described by the International Cosmetic Ingredient Dictionary and Handbook (Dictionary) to function mainly as skin and hair conditioning agents in personal care products.1 This report assesses the safety of the following 19 plant-derived ingredients:

Hydrolyzed Amaranth Hydrolyzed Maple Sycamore Protein Hydrolyzed Avocado Protein Hydrolyzed Pea Protein Hydrolyzed Barley Protein Hydrolyzed Potato Protein Hydrolyzed Brazil Nut Protein Hydrolyzed Sesame Protein Hydrolyzed Cottonseed Protein Hydrolyzed Sweet Almond Protein Hydrolyzed Extensin Hydrolyzed Vegetable Protein Hydrolyzed Hazelnut Protein Hydrolyzed Zein Hydrolyzed Hemp Seed Protein Lupinus Albus Protein Hydrolyzed Jojoba Protein Pisum Sativum (Pea) Protein Hydrolyzed Lupine Protein

The safety of several hydrolyzed proteins as used in cosmetics has been reviewed by the Cosmetic Ingredient Review (CIR) Expert Panel (Panel) in several previous assessments. The Panel concluded that Hydrolyzed Keratin (finalized in 2016), Hydrolyzed Collagen (published in 1985, re-review published in 2006) Hydrolyzed Soy Protein (finalized in 2015), Hydrolyzed Silk (finalized in 2015), Hydrolyzed Rice Protein (published in 2006), and Hydrolyzed Corn Protein (published in 2011) are safe for use in cosmetics.2-8 Additionally, the Panel concluded that Hydrolyzed Wheat Gluten and Hydrolyzed Wheat Protein are safe for use in cosmetics when formulated to restrict peptides to a weight-average MW of 3500 Da or less.9

CHEMISTRY Definition The definitions and functions of the plant-derived protein and peptide ingredients in this report are provided in Table 1. The plant peptides, or plant protein derivatives, form a broad category of materials that are prepared by extraction of proteins from plants and partial hydrolysis to yield cosmetic ingredients.1 Proteins and protein hydrolysates, including those from plants, are used as conditioning agents in hair and skin products.1,10 The preparation of protein hydrolysates can be accomplished via acid (e.g., hydrochloric acid), (e.g., papain hydrolysis) or other methodologies (e.g., steam). The degree of hydrolysis (i.e., how much the proteins are broken down into smaller pieces) may profoundly affect the size and reactivity of such hydrolysates. The degree of hydrolysis can be attenuated by altering the reaction conditions (e.g. change temperature or concentration of the hydrolyzing agents). The ingredients in this report, even those ingredients without “hydrolyzed” in the name, are hydrolyzed to at least some degree as a necessary part of extraction or solubilization.

Physical and Chemical Properties Molecular weights have been provided (by individual suppliers) for several of the plant-derived hydrolyzed proteins; this information is presented in Table 2. A histogram showing the approximate distribution of molecular weights for Hydrolyzed Hazelnut Protein is presented in Figure 1.

Method of Manufacturing Methods used to manufacture protein hydrolysates typically yield broad molecular weight distributions of peptides, ranging from 500 to 30,000 daltons (Da).11 However, certain , such as papain, can routinely yield narrower distributions of 500 to 10,000 Da. For example, if the average molecular weight of an is 135 Da, then, under the broader distribution figures (i.e., 500 to 30,000 Da), these ingredients are approximately 4 to 220 amino acids in length (and approximately 4 to 74 amino acids in length under the narrower distribution, i.e., 500 to 10,000 da).12

Hydrolyzed Amaranth Protein A supplier reported that Hydrolyzed Amaranth Protein (molecular weight (MW = 1500 Da) is produced by filtering a solution of finely ground amaranth powder in water and then reacting the resultant colloidal protein solution with acid for a prescribed period of time and temperature until the hydrolyzed protein solution is obtained.13

Hydrolyzed Avocado Protein Hydrolyzed Avocado Protein is reported to be prepared from sliced and dried avocado fruits.14 Cold pressure is used to extract lipids from the fruits, and then the proteins are hydrolyzed by enzymatic reactions with a cellulase and a . Following centrifugation, the solution is purified by ultrafiltration to remove residual proteins and enzymes. The solution is further purified by nanofiltration to remove salts. The resulting solution is comprised of 20%-50% peptides (w/w) and 20%-30% carbohydrates (w/w).

Hydrolyzed Brazil Nut Protein A supplier reported that Hydrolyzed Brazil Nut Protein is obtained through acid hydrolysis.15

Hydrolyzed Hazelnut Protein A supplier reported that Hydrolyzed Hazelnut Protein is produced by enzymatic hydrolysis.16

Hydrolyzed Lupine Protein A supplier reported that Hydrolyzed Lupine Protein is prepared by hydrolyzing lupine proteins in water through an enzymatic reaction with a protease.17 The solution is then centrifuged and purified by ultrafiltration to remove residual proteins and protease. The solution is further purified by nanofiltration to remove salts.

Hydrolyzed Sweet Almond Protein A supplier reported that Hydrolyzed Sweet Almond Protein is obtained through enzyme hydrolysis.15

Hydrolyzed Vegetable Protein The proteins of corn and soy may be combined and hydrolyzed with enzymes under mild conditions for several hours until the target molecular weight is achieved.18 The resultant hydrolyzed proteins may then be concentrated. This mixture of hydrolyzed corn and hydrolyzed soy protein is sold under the INCI name Hydrolyzed Vegetable Protein. Hydrolyzed vegetable proteins (generic) may also be produced by hydrochloric acid hydrolysis of the proteinaceous by-products of the edible oils or starches of soybean, rapeseed meals, and maize gluten.19

Composition Hydrolyzed Amaranth Protein Unprocessed and extruded amaranth flour were hydrolyzed at a kinetic of 10, 25, 60, 90, 120 and 180 min with the enzymes pepsin and pancreatin, respectively.20 Hydrolysis time impacted the peptide profile, with longer times yielding lower molecular masses of peptides (e.g. unprocessed amaranth hydrolysis yielded molecular masses around 2064 Da at 10 min, 802 Da after 120 min, and 567 Da after 180 min). Extrusion of the amaranth flour yielded more peptides with a lower molecular mass (< 1000 Da) immediately after 10 min of hydrolysis. Hydrolysis with both enzymes produced peptides with biological activity, such as an angiotensin converting enzyme inhibitor (ACE-inhibitor) and a dipeptidyl peptidase 4 inhibitor (DPP-IV inhibitor).

Hydrolyzed Lupine Protein A supplier reported that Hydrolyzed Lupine Protein is comprised of >90% peptides (w/w) and <4% carbohydrates (w/w). 17

Hydrolyzed Vegetable Protein (MSG) occurs naturally in ingredients such as hydrolyzed vegetable protein (generic) and some protein isolates.21 Hydrolyzed vegetable protein may contain 10%-30% MSG.22

Impurities Hydrolyzed Avocado Protein In data supplied by the Personal Care Products Council, Hydrolyzed Avocado Protein has < 0.042 μg/kg benzo[a]pyrene; < 0.10 ppm arsenic; 0.70 ppm barium; <0.10 ppm cadmium; < 0.75 ppm chromium; 0.40 ppm cobalt; < 0.05 ppm lead; and < 0.10 ppm mercury.14 Aflatoxins B&G combined were < 1.86 μg/kg. The absence of organochlorinated, organophosphorylated, and organosulfur compounds from pesticides was noted.

Hydrolyzed Hazelnut Protein A supplier reported that Hydrolyzed Hazelnut Protein has less than 20 ppm heavy metals and less than 2 ppm arsenic.16

Hydrolyzed Lupine Protein In data supplied by the Personal Care Products Council, Hydrolyzed Lupine Protein has < 0.042 μg/kg benzo[a]pyrene; < 0.10 ppm arsenic; 0.70 ppm barium; <0.10 ppm cadmium; < 0.20 ppm chromium; < 0.20 ppm cobalt; < 0.05 ppm lead; and < 0.10 ppm mercury.17 Aflatoxins B&G combined were < 1.86 μg/kg. Organo- pesticide residues were not detected.

Hydrolyzed Vegetable Protein Free and esterified forms of 3-monochloro-1,2-propanediol (3-MCPD) and 1,3-dichloro-2-propanol (1,3- DCP) are reported to be found in acid-hydrolyzed vegetable proteins (generic).23,24 3-MCPD is formed from the reaction of triglycerides in the vegetable protein and hydrochloric acid.19 These are Group 2B compounds (possibly carcinogenic to humans) according to the International Agency for Research on Cancer (IARC).25

USE Cosmetic The safety of the cosmetic ingredients included in this assessment is evaluated based on data received from the U.S. Food and Drug Administration (FDA) and the cosmetics industry on the expected use of these ingredients in cosmetics. Use frequencies of individual ingredients in cosmetics are collected from manufacturers and reported by cosmetic product category in the FDA Voluntary Cosmetic Registration Program (VCRP) database. Use concentration data are submitted by Industry in response to surveys, conducted by the Personal Care Products Council (Council), of maximum reported use concentrations by product category. According to 2016 VCRP data, Hydrolyzed Vegetable Protein is used in 136 formulations; the majority of uses are in leave-on products (Table 3).10 Hydrolyzed Lupine Protein has the second greatest number of overall uses reported, with a total of 103; the majority of the uses are in leave-on formulations. The results of the concentration of use survey conducted in 2016 by the Council indicate Hydrolyzed Potato Protein has the highest reported maximum concentration of use; it is used at up to 2.4% in nighttime skin care products.26 Hydrolyzed Hazelnut Protein is used at up to 0.99% in body and hand skin care products. No uses were reported for Hydrolyzed Avocado Protein, Hydrolyzed Maple Sycamore Protein, or Hydrolyzed Zein in the VCRP or by Council. No uses were reported to the VCRP for Lupinus Albus Protein: this ingredient has not been surveyed by the Council. In some cases, reports of uses were received from the VCRP, but no concentration of use data were provided. For example, Hydrolyzed Cottonseed Protein is reported to be used in 37 formulations, but no use concentration data were provided. In other cases, no uses were reported to the VCRP, but a maximum use concentration was provided in the industry survey. For example, Hydrolyzed Hemp Seed Protein was not reported in the VCRP database to be in use, but the industry survey indicated that it is used at concentrations up to 0.0002%. It should be presumed that Hydrolyzed Hemp Seed Protein is used in at least one cosmetic formulation. Some of these ingredients may be used in products that can come into contact with mucous membranes and the eyes. For example, Hydrolyzed Vegetable Protein is used in bath soaps and detergents at up to 0.25% and Hydrolyzed Jojoba Protein is used in eye makeup preparations at up to 0.0027%.26 Additionally, some of these ingredients were reported to be used in hair sprays, face powders, and fragrances and could possibly be inhaled. For example, Hydrolyzed Hazelnut Protein was reported to be used in perfume at a maximum concentration of 0.25%. In practice, 95% to 99% of the droplets/ particles released from cosmetic sprays have aerodynamic equivalent diameters >10 µm, with propellant sprays yielding a greater fraction of droplets/particles below 10 µm compared with pump sprays.27-30 Therefore, most droplets/ particles incidentally inhaled from cosmetic sprays would be deposited in the nasopharyngeal and bronchial regions and would not be respirable (i.e., they would not enter the lungs) to any appreciable amount.27,29 Conservative estimates of inhalation exposures to respirable particles during the use of loose powder cosmetic products are 400-fold to 1000-fold less than protective regulatory and guidance limits for inert airborne respirable particles in the workplace.31-33 The plant-derived protein and peptide ingredients described in this safety assessment are not restricted from use in any way under the rules governing cosmetic products in the European Union.34

Non-Cosmetic The FDA has defined the term “protein” to mean any α-amino acid polymer with a specific defined sequence that is greater than 40 amino acids in size.35 The FDA considers a “peptide” to be any polymer composed of 40 or fewer amino acids. Hydrolyzed vegetable proteins (generic) are widely used as seasonings and as ingredients in processed savory food products and range in concentration of use in foods from 0.1% to 40%.19,22 Generally, hydrolyzed proteins (acid hydrolyzed or enzymatically hydrolyzed) do not pose a hazard to humans at levels at which they are used as flavoring agents in foods.36 Plant protein products are approved food additives according to the FDA (21CFR§170.3). The FDA requires allergen labeling when major allergens, such as tree nuts, are included in food.37 A major food allergen is an ingredient from a food or food group, such as tree nuts, that contains protein derived from the food.

TOXICOKINETICS No published toxicokinetic studies on plant-derived hydrolyzed proteins and peptides were discovered and no unpublished data were submitted.

TOXICOLOGICAL STUDIES Subchronic Toxicity Studies Lupinus Albus Protein The toxicity of Lupinus albus was studied in a 112-day dietary protein study in Charles River rats.38 Diet consisting of 20% dietary protein from Lupinus albus, Lupinus luteus, or (the control) was fed to groups of 12 animals (sex not reported) ad libitum. The lupine diets were supplemented with DL-methionine. At the end of the experimental period, the animals were killed and the weights of the liver, kidneys, spleen, heart, and adrenals were recorded. Tissue samples of the liver, kidneys, and lungs were examined microscopically. The rats fed the L. albus diets gained weight at a slightly lower rate than those fed L. luteus and casein. There were no differences in the feed intakes and feed efficiencies of both lupine groups during weeks 1-6. There were no differences observed in organ- to-body weight ratios of liver, spleen, heart, and adrenals of rats fed either lupines or casein. No adverse effects were reported. No significant differences were observed in the gross necropsy findings or the microscopic examinations.

GENOTOXICITY No published genotoxicity studies on plant-derived hydrolyzed proteins and peptides were discovered and no unpublished data were submitted.

CARCINOGENICITY No published carcinogenicity studies on plant-derived hydrolyzed proteins and peptides were discovered and no unpublished data were submitted.

OTHER RELEVANT STUDIES Antioxidant Effects Hydrolyzed Hemp Seed Protein No adverse effects were mentioned in a study of rats fed hydrolyzed hemp seed meal protein.39 This study investigated the antioxidant effects of hydrolyzed hemp seed meal protein in spontaneously hypertensive rats. Groups of 8 male rats were fed diets containing 0%, 0.5%, or 1.0% (w/w) hydrolyzed hemp seed meal protein for 8 weeks. Half of the rats were killed for blood collection while the remaining half underwent a 4-week washout and then fed an additional 4 weeks before terminal blood collection. Plasma total antioxidant capacity (TAC), superoxide dismutase (SOD), and catalase (CAT) levels were decreased in the rats that lived through the wash-out phase when compared to those killed prior. Significant (p < 0.05) increases in plasma SOD and CAT levels accompanied by decreases in total peroxide levels were observed in both the pre- and post-wash-out rats. The hemp seed meal protein in this study was hydrolyzed by pepsin and pancreatin, consecutively.

Type 1 Hypersensitivity Hydrolyzed Brazil Nut Protein, Hydrolyzed Hazelnut Protein, Hydrolyzed Sweet Almond Protein As commonly known, tree nuts, including Bertholletia excelsa (Brazil nut), Corylus spp. (hazelnut), and Prunus dulcis (sweet almond) are major food allergens that produce Type 1 (immediate) reactions in sensitized individuals.

Lupinus Albus Protein and Pisum Sativum (Pea) Protein A clinical study examined 3 patients with a history of anaphylactic reactions to peas who subsequently developed signs of sensitization after ingesting peanuts.40 All 3 patients had positive skin prick tests, as well as elevated serum levels of IgE antibodies against protein extracts of both peas and peanuts. IgE-binding experiments revealed strong binding mainly to vicilin in pea extracts and exclusively to Ara h 1 in a crude peanut extract. IgE binding to the purified Ara h 1 of peanuts was definitively inhibited by purified pea vicilin, but the IgE binding to the pea vicilin was not inhibited to any significant extent by peanut Ara h 1. The authors concluded that clinically- relevant cross-reactivity can occur between vicilin homologs in peanuts (i.e., Ara h1) and in peas. They noted that the allergic reactions to peanuts was attributable to cross-reactive IgE antibodies raised previously against pea allergens in these patients, based on the course of the development of allergic reactions, skin prick test results, specific IgE levels, and the potent inhibition by pea vicilin of IgE binding to Ara h1, compared with the lack of inhibition by Ara h 1 of IgE binding to pea vicilin. Immunological cross-reactivity was studied among the seeds of widely different species (lupin and pea, as well as peanut, lentil kidney bean and soybean) using the sera of 12 peanut-sensitive children.42 IgE binding to the seed proteins of these plant species varied widely among the subjects. IgE binding to peanut polypetides was prevalent among the subjects, as expected, but binding to the polypeptides of other legumes was also observed. Often the binding was to the basic subunits of 11S globulins. In this study, the subjects exhibited skin prick test results that generally paralleled the results of the IgE binding studies. The most sensitive subjects had strong reactions to peanut, pea and lentil protein extracts. Skin prick tests were performed using a panel of protein extracts from the seeds of several legume species on patients (n=36) with to peanuts and/or other legumes.43 The plant species tested included lupine and green pea, as well as the dun pea, chick pea, lentil, soybean, white bean and broad bean. The patients were divided into 2 groups. Group 1 included 6 subjects who were not allergic to peanuts but were allergic to lentils (4), dun pea (3), green pea (3), soybean (2), broad bean (2), lupine (1), and/or chickpeas (1). Each of these patients had positive skin prick tests to at least 4 of the legume extracts tested. Group 2 included 30 patients with peanut , and was subdivided into 3 subgroups. Group 2a included 13 patients who were not allergic to other legumes, all of whom exhibited negative skin prick tests to the protein extracts of legumes other than peanuts or ate all legumes other than peanuts without reactions. Group 2b included 8 patients who were sensitized to legumes, in addition to peanuts, without having previously experienced clinical reactions to legumes other than peanuts. These patients exhibited positive skin prick tests to the proteins of at least 1 and up to 5 legumes, in addition to peanut proteins. Group 2c included 9 patients with allergies to peanuts and to other legumes, including green peas (4), dun peas (3), lentils (3), soybeans (2), and lupine (1), and positive skin prick tests to the proteins of at least 1 and up to 5 legumes. In this study, 96% (22/23) of the patients who were sensitized or allergic to legumes other than peanuts (whether or not they were also allergic to peanuts) and 100% (17/17) of the patients allergic to peanuts and other legumes had specific IgE against Ara h 1. Only 54% (7/13) of the patients with peanut allergy who were not also sensitized to other legumes had specific IgE against Ara h 1. Further, peanut protein extracts inhibited the binding of dun pea specific IgE to dun pea proteins. The authors concluded, based on the overall results of their study, that peanut- allergic patients sensitized to Ara h 1 are at greater risk of becoming sensitized or developing allergies to other legumes, compared with those not sensitized to Ara h 1.

DERMAL IRRITATION AND SENSITIZATION STUDIES Irritation 14,16,17,44,45 In vitro and animal dermal irritation studies are presented in Table 4. No irritation was observed in in vitro and animal studies with the following hydrolyzed protein ingredients: amaranth (20% in water), avocado (concentration not reported), hazelnut (100%), lupine (concentration not reported), and vegetable (100%).

Sensitization Animal and human dermal sensitization studies are presented in Table 5.14,16,17,46-48 No sensitization was observed in animal studies of Hydrolyzed Avocado Protein (concentration not reported), Hydrolyzed Hazelnut Protein (up to 100%), and Hydrolyzed Lupine Protein (concentration not reported). No sensitization was observed in human studies of the following hydrolyzed protein ingredients: amaranth (concentration not reported), avocado (concentration not reported), lupine (concentration not reported), potato (up to 2.4% in formulation), and sweet almond (concentration not reported).

Phototoxicity Phototoxicity studies are presented in Table 6.14,16,17 Hydrolyzed Avocado Protein, Hydrolyzed Hazelnut Protein, and Hydrolyzed Lupine Protein were not phototoxic in in vitro studies. The concentrations for these test materials were not reported.

OCULAR IRRITATION STUDIES In vitro and animal ocular irritation studies are presented in Table 7.14,16,17,49,50 In in vitro studies, Hydrolyzed Amaranth Protein (20%) and Hydrolyzed Vegetable Protein (up to 100%) were not irritating, but results were mixed for Hydrolyzed Avocado Protein (concentration not reported), with moderate irritation observed in a hen’s egg test- chorioallantoic membrane (HET-CAM) assay but no severe irritation observed in a bovine cornea opacity permeability (BCOP) test. Hydrolyzed Lupine Protein (concentration not reported) was weakly irritating in both the HET-CAM and BCOP tests. Hydrolyzed Hazelnut Protein was not irritating in animal studies when tested neat.

CLINICAL STUDIES Case Studies Hydrolyzed Vegetable Protein MSG symptom complex has been reported in sensitive people that have consumed foods containing hydrolyzed vegetable protein (generic).22 No adverse effects from cosmetic use were discovered in the published literature.

SUMMARY Hydrolyzed plant-derived proteins and peptides function primarily as skin and hair conditioning agents in personal care products. These protein derivatives are prepared by subjecting vegetable proteins to enzymatic or acid hydrolysis. Hydrolyzed Vegetable Protein has the most reported uses in personal care products, with a total of 136 formulations; the majority of uses are in leave-on products. Hydrolyzed Lupine Protein has the second greatest number of overall uses reported, with a total of 103; the majority of the uses are in leave-on formulations. Hydrolyzed Potato Protein has the highest reported maximum concentration of use; it is used at up to 2.4% in nighttime skin care products. Hydrolyzed Hazelnut Protein is used at up to 0.99% in body and hand skin care products. No uses were reported for Hydrolyzed Avocado Protein, Hydrolyzed Maple Sycamore Protein, or Hydrolyzed Zein in the VCRP or by Council. No uses were reported to the VCRP for Lupinus Albus Protein: this ingredient has not been surveyed yet by the Council. Hydrolyzed vegetable proteins (generic) are widely used as seasonings and as ingredients in processed savory food products and range in concentration of use in foods from 0.1% to 40%. Generally, hydrolyzed proteins (acid hydrolyzed or enzymatically hydrolyzed) at levels used as flavoring agents in foods do not pose a hazard to humans. Plant protein products are approved food additives according to the FDA. The FDA requires allergen labeling when major allergens, such as tree nuts, are included in food. The toxicity of Lupinus albus was studied in a 112-day study in rats where diets were comprised of 20% dietary protein from L. albus, L. luteus, or casein (the control). The rats fed the L. albus diets gained weight at a slightly lower rate than those fed L. luteus and casein. There were no differences in the feed intakes and feed efficiencies of both lupine groups during weeks 1-6. There were no differences observed in organ-to-body weight ratios of liver, spleen, heart, and adrenals of rats fed either lupines or casein. No adverse effects were reported. No significant differences were observed in the gross necropsy findings or the microscopic examinations. No adverse effects were reported in rats fed hydrolyzed hemp seed meal protein. The rats were fed diets containing 0%, 0.5%, or 1.0% (w/w) hydrolyzed hemp seed meal protein for 8 weeks. Tree nuts, including Bertholletia excelsa (Brazil nut), Corylus spp. (hazelnut), and Prunus dulcis (sweet almond) are well known major food allergens that produce Type 1 (immediate) reactions in sensitive individuals. Type 1 allergic responses also have been reported following the consumption of legumes such as peanut, lupine, and pea. In dermal irritation studies, Hydrolyzed Amaranth Protein (20% in water), Hydrolyzed Avocado Protein (concentration not reported), Hydrolyzed Hazelnut Protein (100%), Hydrolyzed Lupine Protein (concentration not reported), and Hydrolyzed Vegetable Protein (100%) were not irritants. No sensitization was observed in animal studies of Hydrolyzed Avocado Protein (concentration not reported), Hydrolyzed Hazelnut Protein (up to 100%), and Hydrolyzed Lupine Protein (concentration not reported). No sensitization was observed in human studies of the following hydrolyzed protein ingredients: amaranth (concentration not reported), avocado (concentration not reported), lupine (concentration not reported), potato (up to 2.4% in formulation), and sweet almond (concentration not reported). Hydrolyzed Avocado Protein, Hydrolyzed Hazelnut Protein, and Hydrolyzed Lupine Protein were not phototoxic in in vitro studies. The concentrations of these test materials were not reported. In in vitro ocular studies, Hydrolyzed Amaranth Protein (20%) and Hydrolyzed Vegetable Protein (up to 100%) were not irritating, but results were mixed for Hydrolyzed Avocado Protein (concentration not reported), with moderate irritation observed in a hen’s egg test- chorioallantoic membrane (HET-CAM) assay but no severe irritation in a bovine cornea opacity permeability (BCOP) test. Hydrolyzed Lupine Protein (concentration not reported) was weakly irritating in both the HET-CAM and BCOP tests. Hydrolyzed Hazelnut Protein was not an ocular irritant in animal studies when tested neat.

DATA NEEDS The majority of the plant proteins and peptides in this assessment are found in foods, and daily exposures from food use would result in much greater systemic exposures than those resulting from use in cosmetic products. The FDA has approved the use of plant protein products as food additives. While some available systemic data has been reported, this safety assessment focuses on the potential for irritation and sensitization from topical exposure to these plant proteins. CIR is seeking dermal and ocular irritation and sensitization data on the 19 plant-derived proteins and peptides at or above the use concentrations described in this report. Additionally, CIR is seeking data on the molecular weight ranges, composition, and impurities. Information on any additional toxicological data that would help the CIR Expert Panel assess the safety of the use of these ingredients in cosmetics and would improve the resulting safety assessment is welcome.

TABLES AND FIGURES

Molecular Weight Distribution for Hydrolyzed Hazelnut Protein (in Daltons) 40%

35%

30%

25%

20%

15%

10%

5%

0% <300 300-510 510-1000 1000-2990 2990-5020 >5020

Figure 1. Molecular weight distribution of Hydrolyzed Hazelnut Protein.16

Table 1. Definitions and functions of the ingredients in this safety assessment.1 (Italicized text below represents additions made by CIR).

Ingredient CAS No. Definition Function Hydrolyzed Amaranth Hydrolyzed Amaranth Protein is the partial hydrolysate of Skin-Conditioning Agent - Misc. Protein amaranth protein derived by acid, enzyme or other method of hydrolysis. Hydrolyzed Avocado Hydrolyzed Avocado Protein is the partial hydrolysate of avocado Skin-Conditioning Agent - Misc. Protein protein derived by acid, enzyme or other method of hydrolysis. Hydrolyzed Barley Protein Hydrolyzed Barley Protein is the partial hydrolysate of barley Hair conditioning Agent; Skin- protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent - Misc. Hydrolyzed Brazil Nut Hydrolyzed Brazil Nut Protein is the partial hydrolysate of brazil Hair conditioning Agent; Skin- Protein nut protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent - Misc. Hydrolyzed Cottonseed Hydrolyzed Cottonseed Protein is the partial hydrolysate of Hair conditioning Agent; Skin- Protein cottonseed protein derived by acid, enzyme or other method of Conditioning Agent - Misc. hydrolysis. Hydrolyzed Extensin Hydrolyzed Extensin is the partial hydrolysate of extensin protein Hair conditioning Agent; Skin- 73049-73-7 derived by acid, enzyme or other method of hydrolysis. Wherein, Conditioning Agent - Misc. extensins are defined as wall-located, basic, hydroxyproline rich structural glycoproteins with alternating hydrophilic and hydrophobic motifs.51 Hydrolyzed Hazelnut Hydrolyzed Hazelnut Protein is the partial hydrolysate of hazelnut Skin-Conditioning Agent - Misc. Protein protein derived by acid, enzyme, or other method of hydrolysis. Hydrolyzed Hemp Seed Hydrolyzed Hemp Seed Protein is the partial hydrolysate of hemp Hair conditioning Agent; Skin- Protein seed protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent - Misc. Hydrolyzed Jojoba Protein Hydrolyzed Jojoba Protein is the partial hydrolysate of jojoba seed Hair conditioning Agent; Skin- 100684-35-3 protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent - Emollient Hydrolyzed Lupine Protein Hydrolyzed Lupine Protein is the partial hydrolysate of lupine Hair Conditioning Agent; Light 73049-73-7 protein derived by acid, enzyme or other method of hydrolysis. Stabilizer; Skin-Conditioning Agent - Misc. Hydrolyzed Maple Hydrolyzed Maple Sycamore Protein is the partial hydrolysate of Hair Conditioning Agent; Skin- Sycamore Protein the protein derived from the maple sycamore tree, Acer Conditioning Agent-Humectant; 73049-73-7 pseudoplatanus, obtained by acid, enzyme, or other method of Skin-Conditioning Agent-Misc. hydrolysis. Hydrolyzed Pea Protein Hydrolyzed Pea Protein is the partial hydrolysate of pea protein Hair Conditioning Agent; Skin- 222400-29-5 derived by acid, enzyme or other method of hydrolysis. Conditioning Agent-Emollient; Skin- 227024-36-4 Conditioning Agent-Misc. Hydrolyzed Potato Protein Hydrolyzed Potato Protein is the partial hydrolysate of potato Hair Conditioning Agent; Skin- 169590-59-4 protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent-Misc. Hydrolyzed Sesame Hydrolyzed Sesame Protein is the partial hydrolysate of sesame Hair Conditioning Agent; Skin- Protein protein derived by acid, enzyme or other method of hydrolysis. Conditioning Agent-Misc. Hydrolyzed Sweet Almond Hydrolyzed Sweet Almond Protein is the partial hydrolysate of Hair Conditioning Agent; Skin- Protein sweet almond protein derived by acid, enzyme or other method of Conditioning Agent-Misc. 100209-19-6 hydrolysis. Hydrolyzed Vegetable Hydrolyzed Vegetable Protein is the partial hydrolysate of Hair Conditioning Agent; Skin- Protein vegetable protein derived by acid, enzyme or other method of Conditioning Agent-Misc. 73049-73-7 hydrolysis. 100209-45-8 Hydrolyzed Zein Hydrolyzed Zein is the partial hydrolysate of Zein derived by acid, Hair Conditioning Agent; Skin- enzyme or other method of hydrolysis. Wherein, Zein is an Conditioning Agent-Misc. alcohol-soluble protein obtained from corn, Zea mays. Lupinus Albus Protein Lupinus Albus Protein is the protein derived from the seeds of Skin-Conditioning Agents – Lupinus albus. Emollient; Skin-Conditioning Agents – Misc. Pisum Sativum (Pea) Pisum Sativum (Pea) Protein is the protein isolated from Pisum Skin-Conditioning Agents – Misc. Protein sativum.

Table 2. Reported molecular weights (Da) of some plant-derived hydrolyzed proteins. Ingredients Value Reference

Hydrolyzed Amaranth Protein ~1500 13

Hydrolyzed Avocado Protein 300-1200 (50-70% w/w) 14

Hydrolyzed Brazil Nut Protein ~150 15

Hydrolyzed Hazelnut Protein 510-1000 (mode value) 16

Hydrolyzed Lupine Protein 300-1200 (40-50% w/w) 17

Hydrolyzed Sweet Almond Protein ~3000 15

Hydrolyzed Vegetable Protein ~1000 52

Table 3. Frequency and concentration of use according to duration and type of exposure for plant-derived hydrolyzed proteins.10,26 # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) Hydrolyzed Amaranth Protein Hydrolyzed Barley Protein Hydrolyzed Brazil Nut Protein Hydrolyzed Cottonseed Protein Totals† 3 0.011 30 0.002 17 0.000026-0.023 37 NR Duration of Use Leave-On 3 0.011 15 NR 7 0.000026-0.016 32 NR Rinse Off NR NR 15 0.002 10 0.000026-0.023 5 NR Diluted for (Bath) Use NR NR NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR NR NR NR NR Incidental Ingestion NR NR NR NR NR NR NR NR Incidental Inhalation-Spray 2; 1a 0.011 1; 12a NR 6a 0.000026; 0.016a 7; 21a; 4b NR Incidental Inhalation-Powder NR NR NR NR NR NR 4b NR Dermal Contact NR NR 1 NR NR NR 37 NR Deodorant (underarm) NR NR NR NR NR NR NR NR Hair - Non-Coloring 3 0.011 29 0.002 14 0.000026-0.023 NR NR Hair-Coloring NR NR NR NR 3 0.00013 NR NR Nail NR NR NR NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR 3 NR Baby Products NR NR 1 NR NR NR NR NR

Hydrolyzed Extensin Hydrolyzed Hazelnut Protein Hydrolyzed Hemp Seed Protein Hydrolyzed Jojoba Protein Totals† 34 0.01-0.13 25 0.25-0.99 NR 0.0002 10 0.001-0.025 Duration of Use Leave-On 29 0.13 24 0.25-0.99 NR NR 4 0.001-0.025 Rinse Off 5 0.01 1 NR NR 0.0002 6 0.001-0.0026 Diluted for (Bath) Use NR NR NR NR NR NR NR NR Exposure Type Eye Area 2 NR 4 NR NR NR 1 0.0027 Incidental Ingestion NR NR NR NR NR NR NR NR Incidental Inhalation-Spray 14a; 3b NR 2a; 18b 0.25 NR NR 3a NR Incidental Inhalation-Powder 3b 0.13c 18b 0.99c NR NR NR 0.001-0.025c Dermal Contact 31 0.13 25 0.25 NR NR 6 0.001-0.0027 Deodorant (underarm) NR NR NR NR NR NR NR NR Hair - Non-Coloring 3 0.01 NR NR NR 0.0002 3 0.001-0.0026 Hair-Coloring NR NR NR NR NR NR NR NR Nail NR NR NR NR NR NR NR NR Mucous Membrane NR NR NR NR NR NR 1 NR Baby Products NR NR NR NR NR NR NR NR

Table 3. Frequency and concentration of use according to duration and type of exposure for plant-derived hydrolyzed proteins.10,26 # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%)

Hydrolyzed Lupine Protein Hydrolyzed Pea Protein Hydrolyzed Potato Protein Hydrolyzed Sesame Protein Totals† 103 NS 5 0.001 12 0.75-2.4 NR 0.001 Duration of Use Leave-On 89 NS 5 0.001 12 0.75-2.4 NR 0.001 Rinse Off 14 NS NR 0.001 NR NR NR 0.001 Diluted for (Bath) Use NR NS NR NR NR NR NR NR Exposure Type Eye Area 17 NS NR NR NR NR NR NR Incidental Ingestion NR NS NR NR NR NR NR NR Incidental Inhalation-Spray 35a; 26b NS 5b NR 9a; 2b NR NR NR Incidental Inhalation-Powder 26b NS 5b 0.001c 2b 0.75c NR 0.001c Dermal Contact 84 NS 5 0.001 12 0.75-2.4 NR 0.001 Deodorant (underarm) NR NS NR NR NR NR NR NR Hair - Non-Coloring 7 NS NR 0.001 NR NR NR 0.001 Hair-Coloring 7 NS NR NR NR NR NR NR Nail 1 NS NR NR NR NR NR NR Mucous Membrane NR NS NR NR NR NR NR NR Baby Products NR NS NR NR NR NR NR NR

Hydrolyzed Sweet Almond Proteind Hydrolyzed Vegetable Protein Pisum Sativum (Pea) Protein Totals† 62 0.001-0.063 136 0.0004-0.3 NR 0.001-0.0025 Duration of Use Leave-On 41 0.001-0.05 70 0.0011-0.3 NR 0.001-0.0025 Rinse Off 21 0.001-0.063 66 0.0004-0.3 NR 0.001-0.0025 Diluted for (Bath) Use NR NR NR NR NR NR Exposure Type Eye Area 9 NR 5 0.0012-0.3 NR NR Incidental Ingestion NR NR NR NR NR NR Incidental Inhalation-Spray 8a; 19b NR 2; 39a; 8b 0.3a NR NR Incidental Inhalation-Powder 19b 0.001-0.05c 1; 8b; 1c 0.0011c NR 0.001c Dermal Contact 39 0.001 53 0.0004-0.3 NR 0.001 Deodorant (underarm) NR NR NR NR NR NR Hair - Non-Coloring 16 0.001-0.063 59 0.0004-0.17 NR 0.001-0.0025 Hair-Coloring 1 NR 24 0.0019 NR NR Nail NR NR NR NR NR NR Mucous Membrane 3 NR NR 0.0004-0.25 NR NR Baby Products NR NR 2 NR NR NR NR = Not reported. NS = Not surveyed. † Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses. a. It is possible these products may be sprays, but it is not specified whether the reported uses are sprays. b. Not specified whether a powder or a spray, so this information is captured for both categories of incidental inhalation. c. It is possible these products may be powders, but it is not specified whether the reported uses are powders. d Two uses for other skin care preparations were categorized under Hydrolyzed Almond Protein in the VCRP. Table 4. Dermal irritation studies for plant-derived hydrolyzed proteins and peptides. Ingredient Concentration Method Results Reference In Vitro Hydrolyzed Amaranth 20% in water EpiDerm MTT Viability assay Non-irritating 44 Protein Hydrolyzed Avocado Not reported 3D human skin model (MTT + Well tolerated 14 Protein IL1α) performed under OECD draft guidelines and ECVAM protocol; 10 volunteers Hydrolyzed Lupine Not reported 3D human skin model (MTT + Well tolerated 17 Protein IL1α) performed under OECD draft guidelines and ECVAM protocol; 20 volunteers Hydrolyzed Vegetable 100% (MW = 750) EpiDerm skin model Non-irritating 45 Protein Animal Hydrolyzed Hazelnut 100% Dermal irritation study performed Non-irritating 16 Protein under OECD Guideline 404; no further details provided

Table 5. Dermal sensitization studies for plant- derived hydrolyzed proteins and peptides. Ingredient Concentration Method Results Reference Animal Hydrolyzed Avocado Not reported LLNA Non-sensitizing 14 Protein Hydrolyzed Hazelnut 100% Guinea pig dermal sensitization study Non-sensitizing 16 Protein performed according to OECD guideline 406 Hydrolyzed Hazelnut 5% Sensitization study using the Marzulli- Non-irritating and non- 16 Protein Maibach method sensitizing Hydrolyzed Lupine Protein Not reported LLNA Non-sensitizing 17 Human Hydrolyzed Amaranth Not reported HRIPT with 108 subjects; semi- No dermal irritation or 47 Protein occlusive sensitization Hydrolyzed Avocado Not reported HRIPT that utilized the Marzulli- Non-sensitizing 14 Protein Maibach method in 50 subjects Hydrolyzed Lupine Protein Not reported HRIPT that utilized the Marzulli- Non-sensitizing 17 Maibach method in 100 subjects Hydrolyzed Potato Protein 1.5% in a face cream HRIPT with 100 subjects; occlusive No dermal irritation or 46 sensitization Hydrolyzed Potato Protein 2.4% in a night cream HRIPT with 100 subjects; occlusive No dermal irritation or 46 sensitization Multiple Hydrolyzed Not reported Sensitization study of protein No adverse reactions to 48 Proteins including hydrolysates in hair care products in 3 Hydrolyzed Sweet Almond Hydrolyzed Sweet Almond groups of patients. Group 1 was Protein were observed. Protein comprised of 11 hairdressers with hand dermatitis, group2 was comprised of 2160 consecutive adults with suspected allergic respiratory disease, and group 3 was comprised of 28 adults with atopic dermatitis. Subjects submitted to scratch and/or prick tests.

Table 6. In vitro phototoxicity studies for plant-derived hydrolyzed proteins and peptides. Ingredient Concentration Method Results Reference Hydrolyzed Avocado Protein Not reported 3T3 Neural Red Uptake (NRU) Not phototoxic 14 method Hydrolyzed Hazelnut Protein Not reported 3T3 NRU method Not phototoxic 16 Hydrolyzed Lupine Protein Not reported 3T3 NRU method Not phototoxic 17

Table 7. Ocular irritation studies for plant-derived hydrolyzed proteins and peptides.

Ingredient Concentration Method Results Reference In Vitro Hydrolyzed Amaranth 20% dilutions EpiOcular MTT viability irritation Non-irritating 49 Protein study Hydrolyzed Avocado Not reported HET-CAM method Moderately irritating 14 Protein Hydrolyzed Avocado Not reported BCOP ocular irritation study Not severely irritating 14 Protein Hydrolyzed Avocado Not reported Neutral red release assay Negligible cytotoxicity 14 Protein Hydrolyzed Lupine Protein Not reported HET-CAM method Weakly irritating 17 Hydrolyzed Lupine Protein Not reported BCOP ocular irritation study Weakly irritating 17 Hydrolyzed Lupine Protein Not reported Neutral red release assay Negligible cytotoxicity 17 Hydrolyzed Vegetable 25%, 50%, and 100% HET-CAM method Practically no irritation potential 50 Protein (MW = 750) Animal Hydrolyzed Hazelnut Neat Ocular irritation study performed Non-irritating 16 Protein under OECD guideline 405; no further details provided

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